The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase

J. D. Potter, J. Gergely

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567 Scopus citations

Abstract

Purified troponin (Tn), the complex of the Ca2+ binding subunit (TnC), the inhibitory subunit (TnI) and the tropomyosin binding subunit (TnT) binds 4 mol of Ca2+ per mol. Two sites bind Ca2+ with a binding constant of 5 x 108 M-1, and 2 with a binding constant of 5 x 106 M-1. In the presence of 2 mM MgCl2, the binding to 4 sites can be characterized with a single affinity constant of 5 x 106 M-1. Purified TnC also binds 4 mol of Ca2+ per mol; 2 sites having a binding constant of 2 x 107 M-1 and 2 have one of 2 x 105 M-1. In the presence of 2 mM MgCl2, the binding constant of the sites of higher affinity is reduced to 2 x 106 M-1, while Ca2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg2+ and Ca2+ for high affinity sites on TnC and Tn, the changes in Ca2+ binding can be accounted for with K(Mg) values of 5 x 103 M-1 and 5 x 104 M-1, respectively. Tn and TnC bind 4 mol of Mg2+ per mol in the absence of Ca2+. The fact that at [Ca2+] 10-5 M four Ca2+ and only two Mg2+ are bound per mol of TnC in the presence of 2 mM Mg2+ further supports the view that there is direct competition between Mg2+ and Ca2+ for the high affinity Ca2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain 6 divalent cation binding sites: 2 high affinity Ca2+ binding sites that also bind Mg2+ competitively (Ca2+ Mg2+ sites); 2 sites with lower affinity for Ca2+ that do not bind Mg2+ (Ca2+ specific sites); and 2 sites that bind Mg2+ but not Ca2+ (Mg2+ specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar adenosine triphosphatase activity as a function of free Ca2+ concentration at 2 different free Mg2+ concentrations suggest that full activation by Ca2+ occurs only upon binding of Ca2+ to the two Ca2+ specific binding sites in Tn but does not require binding of Ca2+ to the Ca2+ Mg2+ sites.

Original languageEnglish (US)
Pages (from-to)4628-4633
Number of pages6
JournalJournal of Biological Chemistry
Volume250
Issue number12
StatePublished - Dec 1 1975

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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